Animals were transcardially perfused with 4% PFA in PBS. Spinal cord and brain tissues were post-fixed with 4% PFA in PBS at 4 °C overnight, cryoprotected in 30% sucrose in PBS, and then embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) for frozen sectioning. Coronal sections were cut at 30-μm thickness on a cryostat and mounted on Matsunami adhesive silane-coated glass slides (Matsunami Glass, Osaka, Japan).
For histology, sections were stained with cresyl violet (Nissl stain; Sigma). Brain lesion volume was estimated by measurement of the area of lost tissue in each of three to five sections spaced 0.5 mm apart. The total lesion volume was calculated as described previously.36 (link)For immunohistochemistry, sections were permeabilized in PBS containing 0.1% X-100 and 0.5% BSA for 1 h at room temperature. Sections were then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. The primary antibodies used were as follows: mouse anti-NeuN (1 : 100; Merck, Cat# MAB377, RRID: AB_11210778), rabbit anti-PHD2 (1 : 200; Abcam), rabbit anti-PKCγ (1 : 100; Santa Cruz, Cat # sc-211, RRID: AB_632234). Alexa Fluor 488- and 588-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies (1 : 500; Invitrogen) were used as secondary antibodies.
For histology, sections were stained with cresyl violet (Nissl stain; Sigma). Brain lesion volume was estimated by measurement of the area of lost tissue in each of three to five sections spaced 0.5 mm apart. The total lesion volume was calculated as described previously.36 (link)For immunohistochemistry, sections were permeabilized in PBS containing 0.1% X-100 and 0.5% BSA for 1 h at room temperature. Sections were then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. The primary antibodies used were as follows: mouse anti-NeuN (1 : 100; Merck, Cat# MAB377, RRID: AB_11210778), rabbit anti-PHD2 (1 : 200; Abcam), rabbit anti-PKCγ (1 : 100; Santa Cruz, Cat # sc-211, RRID: AB_632234). Alexa Fluor 488- and 588-conjugated goat anti-mouse IgG and goat anti-rabbit IgG antibodies (1 : 500; Invitrogen) were used as secondary antibodies.